Establishing the Biosynthetic Pathway of Pteridine Derivatives in Progressive Breast Cancer Project Summary Overview: Pteridine derivatives are metabolic intermediates involved in the biosynthetic pathways of several cofactors and vitamins; however, surprisingly little information is known about their own biosynthetic pathway. In addition to their relevance to folate metabolism, elevated levels of folate-derived pteridines have been reported in the urine of women with breast cancer in a stage-dependent manner. Nevertheless, efforts to characterize their metabolism in human health and disease have been hindered by substantive challenges relating to their quantitative determination under biological conditions. The proposed research will therefore leverage new analytical techniques developed by our team that will for the first time enable the systematic mapping of the pteridine biosynthetic pathway in a clinically relevant progressive breast cancer model. Objectives and Approach: First, we will systematically map the pteridine biosynthetic pathway in a clinically relevant human breast cell line using metabolic flux techniques (Aim #1). Our selection of 15N5-folic acid and 15N5-guanine as stable isotopic tracers will enable straightforward analysis of resulting isotopomer patterns for 15 pteridine derivatives in order to determine pathway connectivity and metabolic flux. Next, we will examine the tumorous origin of elevated pteridine levels reported in women with breast cancer using an isogenic series of sequentially derived human breast cell lines that progress to a malignant phenotype (Aim #2). Our selection of an in vitro disease model will eliminate possible systematic factors, such as activated immune responses, providing us with critical insights into how pteridine metabolism may be affected by breast cancer progression. Anticipated Findings and Potential Impacts: Current understanding on the prevalence and role of folate-derived pteridines in human health and disease is limited. The proposed research aims squarely at developing a generalizable approach to the study of pteridine metabolism, which is likely to be explored by other research teams examining pteridines in different biological and clinical contexts. Moreover, the research addresses a major gap in our understanding on the relationship between folate-derived pteridines and cancer metabolism. Specifically, the association between pteridine levels and breast cancer invasiveness in our disease model will provide key insights into the possible mechanisms that lead to elevated pteridine levels in cancer patients, which will be explored in a later R01 grant proposal. Ultimately, this information could inform diagnosis and staging of different cancers and identify new targets for the control of tumor formation and growth.